Selectively Isolation and Molecular Identification by dnaJ Gene of Legionella pneumophila Found in Various Water Sources in Dhaka, Bangladesh

Abstract

BACKGROUND: Legionella spp. are the etiological agents of Legionnaires’ disease, often severe pneumonia. After the first severe outbreak of Legionella spp., it was found widely dispersed in natural water sources and humid environments and has become a concern for these sources’ water quality and safety. The fecal indicators used for water quality assessment do not predict the presence of these pathogens; thus, in the absence of targeted analysis, the risk is unobserved until the emergence of infections. There is no study on the presence of Legionella in unattended water bodies in Bangladesh, such as swimming pools, air-conditioned cooling water, and stagnant circulatory water systems. METHODS: A total of 42 water samples were collected from 12 different sites in Dhaka, Bangladesh. Acid, heat, and chlorine treatment were applied to reduce the load of non-Legionellae in water samples. Chlorine treatment was used to kill unwanted organisms in water samples. After successful pretreatment, samples were cultured on buffered charcoal yeast extract agar (BCYE) media. Optimum pH, concentrations of chlorine, and temperature were set up by extensive trial and error culturing of Legionella on BCYE media to minimize other bacterial growth and maximize Legionella growth. Isolated colonies were confirmed primarily by morphological characteristics on BCYE media. Isolates were then confirmed by polymerase chain reaction and gel electrophoresis techniques. We detected the presence of 16S rDNA for Legionella spp. and the dnaJ gene for Legionella pneumophila species identification. Antibiotic sensitivity tests (minimum inhibitory concentration) were also conducted to see if it was alarming for our community’s health. RESULTS: Most of the Legionella spp. could grow at various temperatures, such as 45°C, 50°C, and 55°C, and they could also survive at the normal levels of chlorination, such as 0.25 mg/L and 0.5 mg/L. We have found that, out of 12 different sites, 4 out of 5 (80%) isolates from cooling tower water, 1 out of 4 (25%) from the swimming pool, and 1 out of 3 (33%) household stagnant water are contaminated with Legionella. However, 3 out of 5 isolates found are L. pneumophila from cooling water sites. Furthermore, strains of Legionella spp. were resistant to many antibiotics, such as ciprofloxacin, azithromycin, and tetracycline. CONCLUSIONS: Serious and fatal Legionella spp. (L. pneumophila) infections may be transmitted because of the widespread presence of these water sources and the large number of users in the community. This threat is worsened by the survival of Legionella under various commonly used antibiotic conditions and further exacerbated by their antibiotic resistance. Health-care professionals may face great challenges due to the lack of studies about its prevalence and treatment procedures.